Methods and compositions for healing and repair of articular cartilage

ABSTRACT

Methods and compositions are provided for the treatment of articular cartilage defects and disease involving the combination of tissue, such as osteochondral grafts, with active growth factor. The active growth factor is preferably a composition containing at least one bone morphogenetic protein and a suitable carrier. The method results in the regeneration of functional repair of articular cartilage tissue.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuation of U.S. patent application No. 11/928,977, filedOct. 30, 2007, which is a divisional of U.S. patent application Ser. No.10/779,638, filed Feb. 18, 2004, now U.S. Pat. No. 7,323,445, which is acontinuation of U.S. patent application Ser. No. 09/493,545, filed Jan.28, 2000, now U.S. Pat. No. 6,727,224, which claims priority from U.S.Provisional Patent Application No. 60/118,160, filed Feb. 1, 1999, allof which are incorporated herein by reference.

DESCRIPTION OF THE INVENTION

1. Field of the Invention

The present invention relates to the field of tissue repair,specifically, the regeneration of stable and functional articularcartilage repair. Thus, the present invention may be useful inreconstructive surgery or other procedures for the regeneration orrepair of articular cartilage.

2. Background of the Invention

The repair of articular cartilage injuries remains a challenge inpresent day orthopedics. Several of the current therapeutic strategiesare based upon the grafting of chondral and osteochondral tissues.Autologous osteochondral grafting provides the most appropriatephysiological material. However, donor tissue is limited, and oftenrequires surgery at a secondary site in order to harvest tissue fortransplant. Accordingly, despite substantial endeavors in this field,there remains a need for an effective method of repair of articularcartilage defects and injuries which provides appropriate physiologicalrepair without the need to collect autologous tissue from the patient.

SUMMARY OF THE INVENTION

The present invention provides methods and compositions for regeneratingfunctional and physiologically appropriate tissue repair for the repairof articular cartilage injuries and defects. In particular, the presentinvention comprises methods of treating patients with articularcartilage injuries or defects. The methods and compositions of thepresent invention are advantageous in that they utilize bonemorphogenetic proteins (BMPs), which are known to have osteogenic and/orchondrogenic properties, and which may be produced via recombinant DNAtechnology, and therefore are of potentially unlimited supply. Themethods and compositions of the present invention are furtheradvantageous in that regeneration of functional articular cartilage maybe accelerated or may be of greater ultimate strength and stability, andthe tissue formed at the site of the defect or injury is physiologicallyappropriate.

The use of BMP to augment the repair of articular cartilage defects andinjuries may result in better methods for treatment of osteoarthritis,thus obviating, delaying or reducing the need for artificial hipreplacements and other common interventions. Preclinical evaluationsindicate that rhBMP-2 improves early healing of full thickness defectsof articular cartilage in rabbits.

DETAILED DESCRIPTION OF THE INVENTION

According to the present invention, methods and compositions areprovided for treatment of patients who suffer from some form ofarticular cartilage injury or defect. The injury may be the result ofacute stress, or injury, such as resulting from participation inathletics, or from accidental occurrences which tear, mar or otherwiseinjure the articular cartilage.

The methods and composition are advantageous in that repair orimprovement of articular cartilage defects, particularly full thicknessarticular cartilage defects. Other defects may also be treated by themethods and compositions of the present invention, particularly with anadditional procedure in which the site of the defect is furtheraggravated so as to reach the underlying subchondral bone.

In the present invention, active growth factor, such as a BMP, is addedto a suitable tissue source. The tissue source may be an osteochondralgraft, either autologous to the patient, or may comprise allograft orartificially prepared tissue. In a preferred embodiment, the tissuesource may be chondrocytic cell cultures, such as chondrocyte or stemcell cultures which have been prepared through ex vivo cell culturemethods, with or without additional growth factors. For example, see thedisclosure of U.S. Pat. No. 5,226,914; U.S. Pat. No. 5,811,094; U.S.Pat. No. 5,053,050; U.S. Pat. No. 5,486,359; U.S. Pat. No. 5,786,217 andU.S. Pat. No. 5,723,331. The disclosures of all of these applicationsare hereby incorporated herein by reference.

The tissue may also be harvested by traditional non-cell culture basedmeans, using techniques such as mosaicplasty, in which cartilage isharvested using commercially available instruments such as Acufex7[Smith and Nephew, Inc., Andover Mass.]; COR System [innovasiveTechnologies, Marlborough Mass.]; or Arthrex7 Osteochondral AutograftTransfer System [Arthrex, Munich, Germany]. The tissue harvested may beapplied directly in the methods of the present invention, or may becombined with the tissue based cell culture systems described above.

Growth Factor

The active growth factor used in the present invention is preferablyfrom the subclass of proteins known generally as bone morphogeneticproteins (BMPs), which have been disclosed to have osteogenic,chondrogenic and other growth and differentiation type activities. TheseBMPs include rhBMP-2, rhBMP-3, rhBMP-4 (also referred to as rhBMP-2B),rhBMP-5, rhBMP-6, rhBMP-7 (rhOP-1), rhBMP-8, rhBMP-9, rhBMP-12,rhBMP-13, rhBMP-15, rhBMP-16, rhBMP-17, rhBMP-18, rhGDF-1, rhGDF-3,rhGDF-5, rhGDF-6, rhGDF-7, rhGDF-8, rhGDF-9, rhGDF-10, rhGDF-11,rhGDF-12, rhGDF-14. For example, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6 andBMP-7, disclosed in U.S. Pat. Nos. 5,108,922; 5,013,649; 5,116,738;5,106,748; 5,187,076; and 5,141,905; BMP-8, disclosed in PCT publicationW091/18098; and BMP-9, disclosed in PCT publication W093/00432, BMP-10,disclosed in U.S. Pat. No. 5,637,480; BMP-11, disclosed in U.S. Pat. No.5,639,638, or BMP-12 or BMP-13, disclosed in U.S. Pat. No. 5,658,882,BMP-15, disclosed in U.S. Pat. No. 5,635,372 and BMP-16, disclosed inU.S. Pat. No. 5,965,403. Other compositions which may also be usefulinclude Vgr-2, and any of the growth and differentiation factors [GDFs],including those described in PCT applications WO94/15965; WO94/15949;WO95/01801; WO95/01802; WO94/21681; WO94/15966; WO95/10539; WO96/01845;WO96/02559 and others. Also useful in the present invention may be BIP,disclosed in WO94/01557; HP00269, disclosed in JP Publication number:7-250688; and MP52, disclosed in PCT application WO93/16099. Thedisclosures of all of these applications are hereby incorporated hereinby reference. Also useful in the present invention are heterodimers ofthe above and modified proteins or partial deletion products thereof.These proteins can be used individually or in mixtures of two or more,and rhBMP-2 is preferred.

The BMP may be recombinantly produced, or purified from a proteincomposition. The BMP may be homodimeric, or may be heterodimeric withother BMPs (e.g., a heterodimer composed of one monomer each of BMP-2and BMP-6) or with other members of the TGF-β superfamily, such asactivins, inhibins and TGF-β1 (e.g., a heterodimer composed of onemonomer each of a BMP and a related member of the TGF-β superfamily).Examples of such heterodimeric proteins are described for example inPublished PCT Patent Application WO 93/09229, the specification of whichis hereby incorporated herein by reference. The amount of osteogenicprotein useful herein is that amount effective to stimulate increasedosteogenic activity of infiltrating progenitor cells, and will dependupon the size and nature of the defect being treated, as well as thecarrier being employed. Generally, the amount of protein to be deliveredis in a range of from about 0.05 to about 1.5 mg.

In a preferred embodiment, the osteogenic protein is administeredtogether with an effective amount of a protein which is able to inducethe formation of tendon- or ligament-like tissue. Such proteins, includeBMP-12, BMP-13, and other members of the BMP-12 subfamily, as well asMP52. These proteins and their use for regeneration of tendon andligament-like tissue are disclosed in U.S. application Ser. No.08/362,670, filed on Dec. 22,1994, the disclosure of which is herebyincorporated herein by reference. In another preferred embodiment, aheterodimer in which one monomer unit is an osteogenic protein such asBMP-2, and the other monomer subunit is a tendon-inducing protein, suchas BMP-12, is administered in accordance with the methods describedbelow, in order to induce the formation of a functional attachmentbetween connective tissue and bone.

Application Of Growth Factor

Growth factor may be applied to the tissue source in the form of abuffer solution. One preferred buffer solution is a compositioncomprising, in addition to the active growth factor, about 1.0 to about10.0% (w/v) glycine, about 0.1 to about 5.0% (w/v) of a sugar,preferably sucrose, about 1 to about 20 mM glutamic acid hydrochloride,and optionally about 0.01 to about 0.1% of a non-ionic surfactant, suchas polysorbate 80. Preferred solutions are from about 1% to about 20%w/v cellulosic carrier/buffer. If desired, a salt may be added.

Other materials which may be suitable for use in application of thegrowth factors in the methods and compositions of the present inventioninclude hyaluronic acid, surgical mesh or sutures, polyglyconate,temperature-sensitive polymers, demineralized bone, minerals andceramics, such as calcium phosphates, hydroxyapatite, etc., as well ascombinations of the above described materials. In the preferredembodiment of the present invention, however, no carrier is employed.

The growth factor of the present invention, in a suitable buffer such asthat described above, or combined with a suitable carrier, may beapplied directly to the tissue and/or to the site in need of tissuerepair. For example, the growth factor may be physically applied to thetissue through spraying or dipping, or using a brush or other suitableapplicator, such as a syringe for injection. Alternatively, or inconjunction, the protein may be directly applied to the site in need oftissue repair.

The following examples further describe the practice of embodiments ofthe invention with BMP-2. The examples are not limiting, and as will beappreciated by those skilled in the art, can be varied in accordancewith the above specification.

EXAMPLES I. Rabbit Allograft:

All procedures were carried out with approval from IACUC. Twelve maleNew Zealand white rabbits (6 months old) were used. Two rabbits servedas donors and 10 as recipients. Osteochondral grafts (3.5 mm diameter)were harvested from the trochlear groove or the medial femoral condyleof the donors, and transplanted into a 3.5 mm deep defect in thetrochlear groove of the recipient. The graft was bathed in eitherrhBMP-2 (0.5 mg/ml) or buffer control prior to implantation. The rabbitswere sacrificed 4 weeks after surgery and the transplants andsurrounding tissue were evaluated by a histologic-histochemical gradingscale, as described in Sellers et al., J. Bone Joint Surg., 79-A:1452-1463 (1997). Computerized image analysis of histologic sections wasalso performed. Results were evaluated using the unpaired Studentst-test.

On gross examination, the joints showed no signs of inflammation. Allthe defects were filled by repair tissue. The surface appearance of thedefects was variable but acceptable and did not correlate with form oftreatment. Osteophytes were found in 3 joints (2 in the experimentalgroup; 1 in control buffer group).

There was no correlation between the gross and histologic appearance inany of the defects. The presence of chondrocytes in the lacunae andsporadic cloning of cells in the donor cartilage indicated survival ofthe tissue. Focal degeneration of the donor cartilage was present in allof the control groups, but only one of the rhBMP-2 treated group. Thehealing of the defect in the rhBMP-2 treated group was significantlyimproved compared to that in the control group. The rhBMP-2 treatedgroup had improved bony integration indicated by less fibrous repairtissue in the subchondral bone compartment. Treatment with rhBMP-2 alsoresulted in more cartilage above the original tidemark, apparentlyconsisting of both donor tissue and newly regenerated recipientcartilage. There was no significant difference in the total amount ofbone observed between the two groups.

TABLE I HISTOLOGIC SCORE AND HISTOMORPHOMETRIC MEASUREMENT FOR CARTILAGEREPAIR, MEAN VALUE (SD) Parameter rhBMP-2 Control Average Score** 10.0(5.42)* 20.6 (5.18) % of bone under tidemark 73.26 (13.28) 62.88 (18.07)% of fibrous tissue under tidemark  2.19 (2.04)* 15.81 (9.88) % ofcartilage above tidemark 74.70 (41.08)* 18.17 (26.70) % of filing of thedefect 96.53 (4.86)* 88.79 (8.04) *Statistically significant differencefrom control (p < 0.05). **Scale system ranges from 0 (normal cartilage)to 31 (no repair).

Additional histomorphometric analysis data further supports thebeneficial effects of rhBMP-2 on the healing of graft. For example, thepercentage filling of the new tissue above tide marker has been shown tobe 81.52% in a rhBMP-2 treated group vs. 57.63% in control. There wasless graft cartilage degeneration in rhBMP-2 treated group (23.83%) thanin control group (44.52%). The integration of the graft or newly formedcartilage with the host cartilage was improved by rhBMP-2 treatment(56.48%) compared to that of control group (21.89%). More new cartilageformed under the influence of rhBMP-2 either at the edge of graft, whicheliminated the gap between the graft and host, or at the top of graft,which made the graft more congruent with the joint surface.

The above results demonstrate that the healing of allogeneicosteochondral grafts in articular cartilage defects was improved by theaddition of rhBMP-2. The active growth factor may have acceleratedsubchondral bone union, providing support and nutrition to the articularcartilage tissue. Addition of growth factor may also have stimulated newcartilage formation from recipient mesenchymal stem cells in the bonemarrow and/or the synovial tissue. These results suggest that thecombination of active growth factor, particularly the bone morphogeneticproteins, and osteochondral allografts might present a potent strategyfor treatment of articular cartilage defects, particularly fullthickness articular cartilage defects.

II. Rabbit Autograft:

Osteochondral grafts (2.7 mm in diameter and 3.0 mm long) were harvestedfrom the trochlear groove or femoral condyle and transplanted into adonor site 2.7mm wide and 3.5mm long on the trochlear groove or femoralcondyle of the knee joint in rabbits. Half the animals had bufferdripped into the recipient site prior to transplantation, and then thegrafts were dipped in buffer for 2 minutes and placed into the recipientsite. The other half had 5 μg rhBMP-2 dripped into the recipient siteprior to transplantation, and then the graft was dipped into buffercontaining 500 μg/ml rhBMP-2 for 2 minutes and then transplanted intothe recipient site. The animals were sacrificed 4 weeks after surgery,and the recipient sites were evaluated histologically using both ahistologic-histochemical grading scale [Sellers, et al., J. Bone JointSurg., 79-A: 1452-63 (1997)] and quantitative computerized imageanalysis of the tissue. The data indicated that treatment with rhBMP-2improved the healing of the autograft. The most dramatic effects werethe reduction of graft cartilage degeneration (rhBMP-2 8.18% vs. control36.25%), and more cartilage formed at the edge of graft (rhBMP-2 88.23%vs. control 50%).)

III. Non-Human Primate Autograft:

The non human primates used for autografts experiments were cynomologousmacaques. Osteochondral grafts (3.5 mm diameter×6 mm long) wereharvested from the trochlear groove of 6 cynomologous macaques andtransplanted into recipient sites drilled into both the medial andlateral femoral condyle of the same animal (n=12 transplants total).Prior to transplantation 25 μg rhBMP-2 was dripped into 6 recipientsites, and the grafts from those 6 transplants were dipped into asolution of 1.25 mg/ml rhBMP-2 for 2 minutes. In the other 6transplants, buffer alone was dripped into the recipient sites and thegrafts were dipped into buffer alone for 2 minutes prior totransplantation. The limbs were immobilized in a cast for 2 weekspost-operatively, and the animals were sacrificed 9 weeks postoperatively.

All the animals had normal function of their knee joints. On grossexamination, the joints showed no signs of inflammation. Osteophyteswere not found in any joint. Although the surface of the defectsappeared level with the surrounding cartilage on gross examination,microscopic observation revealed subsidence of the grafts in most of thecases. The tissue observed grossly covering the surface was actuallynew-formed tissue on the top of graft. Computerized image analysis wasperformed by a blinded evaluator to quantitate percent filling of thedefect, the new tissue types formed above the original tide mark, andthe integration of the grafts and the surrounding cartilage. Favorableresults were observed in the rhBMP-2 treated group in all theseparameters. More new cartilage formed between the graft and hostcartilage to eliminate the gap resulting in better integration of thegraft with the surrounding cartilage (rhBMP-2 88.59% vs. control64.82%). The filling of the cartilage defect was better in rhBMP-2treated group (95.02%) than in the control group (86.68%). There wasmore fibrous tissue in the control group (11.90% vs. rhBMP-2 5.65%),while more transitional tissue was found in the rhBMP-2 treated group(36.38% vs. control 20.53%). There was no significant difference on theoverall histologic-histochemical score between the two groups.Peripheral quantitative computered tomography (PQCT) showed that thebone density increased in the donor sites with time. At 6 weeks and 9weeks after the operation, the tissue in the rhBMP-2 treated donor siteswas significantly denser and the healing process was more advancedcompared to control sites. Histologically, the donor sites containedregenerated bone trabeculae with fibrous tissue at the surface in allthe cases.

IV. rhBMP-2 Retention Ex Vivo:

Retention of rhBMP-2 in osteochondral graft with this technique wasevaluated with the grafts from non-human primates. The graft was dippedin a mixture solution of 1251 labeled rhBMP-2 and unlabeled rhBMP-2.Results showed that the amount of rhBMP-2 absorbed to graft wasproportional to the concentration of the protein, and the time ofsoaking. Other factors, which affect the retention of rhBMP-2, includedthe size of graft, and the presence of marrow elements betweentrabecular bone.

V. rhBMP-2 Retention Time Course In Vivo:

The time course of rhBMP-2 retention in osteochondral graft wasevaluated in rabbits. A mixture solution of ¹²⁵I labeled rhBMP-2 andunlabeled rhBMP-2, which contained 5 μg rhBMP-2 and 20 μCi ¹²⁵I, wasloaded to the graft before implantation. The animals were scanned withy-camera during the follow-up time for 22 days post-operatively.Compared to the time course of collagen sponge as a carrier, the halftime of rhBMP-2 in osteochondral graft was increased from 1 day to 3days. The radioactivity of 10% of the starting point was maintained from11 days of collagen sponge to 22 days of graft.

VI. Non-Human Primate Allografts:

Donor sites (3.5 mm wide×6 mm long) were removed from the trochleargrooves of 12 adult cynomologous macaques and transplanted into 3.5×mmrecipient sites in the medial and lateral femoral condyles of unrelatedindividuals. Half of the transplants were soaked in 1.25 mg/ml rhBMP-2for 2 minutes prior to transplantation, and half were soaked in buffer.The identical procedure was performed on the other limb 7 weeks afterthe first surgery. The limb was immobilized in a cast for 2 weeks postoperatively after each surgery, and the animals were sacrificed 9 weeksafter the second surgery for histologic analysis.

These results suggest that the combination of active growth factor,particularly the bone morphogenetic proteins, and osteochondralautografts might present a potent strategy for treatment of articularcartilage defects, particularly full thickness articular cartilagedefects.

In other embodiments BMP-2 may also be applied to frozen osteochondralallograft for treatment of focal articular cartilage defect.

The foregoing descriptions detail presently preferred embodiments of thepresent invention. Numerous modifications and variations in practicethereof are expected to occur to those skilled in the art uponconsideration of these descriptions. Those modifications and variationsare believed to be encompassed within the claims appended hereto.

1. A method for treating a full thickness defect of articular cartilagecomprising administering to the area of the defect an osteochondralgraft having applied thereto a composition consisting essentially of atleast one purified bone morphogenetic protein (BMP) in an amounteffective for the regeneration of articular cartilage.
 2. The method ofclaim 1, wherein the graft is an autograft.
 3. The method of claim 2,wherein the autograft is obtained by mosaicplasty.
 4. The method ofclaim 1, wherein the graft is an allograft.
 5. The method of claim 1,wherein the BMP is selected from the group consisting of BMP-2, BMP-4,BMP-6, BMP-7, BMP-12, BMP-13, and heterodimers and mixtures thereof. 6.The method of claim 5, further comprising administering to the area inneed of articular cartilage regeneration a protein that induces theformation of tendon or ligament tissue selected from the groupconsisting of BMP-12, BMP-13, MP-52, and heterodimers and/or mixturesthereof.
 7. The method of claim 1, wherein the BMP is BMP-2.
 8. Themethod of claim 1, wherein the BMP is a heterodimer, comprising onesubunit of BMP-2, and one subunit of BMP-6.
 9. A method for treating afull thickness defect of articular cartilage comprising administering tothe area of the defect an osteochondral graft having applied thereto acomposition consisting essentially of: (i) at least one purified bonemorphogenetic protein (BMP) selected from the group consisting of BMP-2,4, 5, 6, 7, and heterodimers and mixtures thereof in an amount effectivefor the regeneration of articular cartilage; and (ii) at least onepharmaceutical carrier selected from the group consisting of hyaluronicacid, surgical mesh or sutures, polyglyconate, temperature-sensitivepolymers, demineralized bone, mineral, calcium phosphate, a ceramic,hydroxyapatite, and combinations thereof.
 10. A method for treating afull thickness defect of articular cartilage comprising administering tothe area of the defect an osteochondral graft and a compositionconsisting essentially of at least one purified bone morphogeneticprotein (BMP) selected from the group consisting of BMP-2, 4, 5, 6, 7,and heterodimers and mixtures thereof, in an amount effective for theregeneration of articular cartilage, wherein said composition is applieddirectly to the osteochondral graft and/or administered directly to thesite in need of tissue repair in conjunction with the graft.
 11. Amethod for treating a full thickness defect of articular cartilagecomprising administering to the area of the defect an osteochondralgraft having applied thereto a composition consisting essentially of atleast one purified bone morphogenetic protein (BMP) selected from thegroup consisting of BMP-2, 4, 5, 6, 7, and heterodimers and mixturesthereof, in an amount effective for the regeneration of articularcartilage, wherein the area in need of regeneration of articularcartilage is selected from the group consisting of the hip and the knee.12. A method for treating a full thickness defect of articular cartilagecomprising administering to the area of the defect an osteochondralgraft having applied thereto a composition consisting essentially of aheterodimeric protein in an amount effective for the regeneration ofarticular cartilage, wherein the heterodimeric protein comprises onepurified bone morphogenetic protein (BMP) selected from the groupconsisting of BMP-2, 4, 5, 6, and 7, and one protein which induces theformation of tendon or ligament tissue selected from the groupconsisting of BMP-12, BMP-13, and MP-52.